How DNA polymorphisms result in variation in complex phenotypes remains a key question in biology and medicine. Recent assays of regulatory variation suggest that the majority of phenotypic deviations are caused by alterations in gene expression. Transcript initiation, production, and stability are affected by DNA polymorphisms (expression polymorphisms, EPs) affecting transcript level, with approximately a half of all alterations due to EPs in cis-regulatory regions of genes. Are there many small-effect or few large- contribution EPs? Are they mostly in modules known to affect transcription or in intermodule regions? Are EP effects additive or epistatic, and how much do they interact with trans polymorphisms in transcription factors? Are they in conserved regions or those evolving quickly between species? What are the effects of EPs segregating in natural populations on organismal performance? Are they neutral, slightly deleterious, or beneficial in some ecological settings or genetic backgrounds? These questions will be answered by resequencing regulatory and coding regions often genes - known to genetically vary for transcript level, and best studied for transcription regulation - in 200 natural isogenic lines of Drosophila melanogaster. Allele- specific expression of the above 200 naturally varying alleles in heterozygous flies will be measured in comparison to common standard alleles using RNA from whole bodies or selected tissues. This will distill cis- influences only while greatly reducing environmental effects and eliminating noise from trans- factors. Association tests will be used to identify EPs affecting transcript level. If cis- by trans- interactions are widespread, chromosomal substitutions will be employed to make the genetic background equivalent among lines. To confirm the function of candidate EPs, 1920 unrelated genotypes will be additionally genotyped and phenotyped for candidate EPs. Such a large data set will enable modeling of expression in terms of all the segregating polymorphisms and interactions between them. Effects of variations in trans- factors, and their interactions will also be evaluated. For the genes bound by transcription factors (TF) with available knockouts, quantitative complementation tests are proposed to study interactions between EPs and polymorphisms in TFs. These studies will contribute to building a pathway-minded description of complex character variation. They will shed light on the genetic architecture of transcript level variation in nature.